215 LIVE PIGLETS GENERATED BY SOMATIC CELL NUCLEAR TRANSFER FOLLOWING TARGETING OF A PORCINE ENHANCED GREEN FLUORESCENT PROTEIN LINE MEDIATED BY ZINC-FINGER NUCLEASES TO ESTABLISH CLONED HYGROMYCIN-RESISTANT PRIMARY CELL LINES SUITABLE FOR Cre-MEDIATED RECOMBINASE-MEDIATED CASSETTE EXCHANGE

Recently, site-specific nucleases (zinc-finger nucleases, ZFN; TAL effector nucleases; and CRISPR) emerged as powerful tools for gene modification of different cells types and enhanced green fluorescent protein (EGFP)-specific ZFN were successfully used in the rat (Geurtz et al. 2010) and in the pig (Watanabe et al. 2010; Whyte et al. 2010). Previously (Brunetti et al. 2008 Clon. Stem Cells), we generated an EGFP transgenic porcine line (Verro2GFP) characterised by a single integration of pCAGGS-EGFP cassette, high ubiquitous EGFP expression, Mendelian transgene transmission, and expression in F1. The aim of this work was to modify a transcriptionally active GFP-locus into one suitable for Cre-mediated recombinase-mediated cassette exchange (RMCE), using EGFP-specific ZFN. Homology arms for promoter-less targeting vector were derived from pCAGGS-EGFP vector (promoter fragment = left-homology-arm = LHA; polyA sequence = right-homology-arm = RHA). Cloning floxed (lox2272/lox5171) hygromycin resistance coding sequence between LHA and RHA sequences, we generated the targeting/RMCE vector (pB5′3′Hygro-PL) and its positive control (C+) for PCR set-up (100–1000 plasmid copies). Verro2GFP fibroblasts cultured in DMEM+M199(1 : 1) + 10% FCS, bFGF in 5% CO2, 5% O2, were transfected using Nucleofector (V-024 program). In ZFN-mediated gene targeting, 2 μg of each ZFN coding vector (Sigma-CompoZr®) and 2 μg of pB5′3′Hygro-PL/KpnI vector were used to “nucleofect” 1.4 × 106 Verro2GFP fibroblasts in 2 experiments. Transfected cells were plated in 20 Petri dishes (Ø = 150 mm) and cultured under hygromycin selection (200 μg mL–1) for 15 days. After 12 days of drug selection, 82 resistant colonies were picked up and expanded in 24 multiwell plates for SCNT. All colonies were PCR screened and 45 (54.9%) colonies were positive. Four colonies were used in zona-free SCNT experiments with 140 Day 6 compacted morulae/blastocysts transferred into 2 synchronized sows that both became pregnant. One pregnancy went to term and delivered 5 live animals and 5 stillborn with correct hygromycin cassette integration, detected by PCR. The PCR products were sequenced in 7 animals to verify integration of promoterless targeting vector and in all 7 sequenced samples we obtained a correct insertion without any substitution/deletion. Using hygromycin selection in these experiments, we demonstrated that ZFN-mediated gene targeting can be easily done with high efficiency and is compatible with living animals. Moreover, we have validated a feasible SCNT-tested platform for further Cre-mediated site-specific gene modifications.